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Yeast histone H3 lysine 4 demethylase Jhd2 regulates mitotic rDNA condensation.

Ryu HY, Ahn S - BMC Biol. (2014)

Bottom Line: We found that Lys4, Lys79, and Lys36 methylation within histone H3 acts as a bivalent marker for the regulation of rDNA recombination and RENT complex-mediated rDNA silencing, both of which are Sir2-dependent pathways.By contrast, we found that Jhd2, an evolutionarily conserved JARID1 family H3 Lys4 demethylase, effects all states of methylated H3K4 within the NTS regions of rDNA and that its activity is required for the regulation of rDNA silencing in a Sir2-independent manner.Our results show that yeast Jhd2, which demethylates histone H3 Lys4 near the rDNA locus, regulates rDNA repeat stability and rDNA silencing in a Sir2-independent manner by maintaining Csm1/Lrs4 and condensin association with rDNA regions during mitosis.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Nucleolar rDNA is tightly associated with silent heterochromatin, which is important for rDNA stability, nucleolar integration, and cellular senescence. Two pathways have been described that lead to rDNA silencing in yeast: 1) the RENT (regulator of nucleolar silencing and telophase exit) complex, which is composed of Net1, Sir2, and Cdc14 and is required for Sir2-dependent rDNA silencing; and 2) the Sir2-independent silencing mechanism, which involves the Tof2 and Tof2-copurified complex, made up of Lrs4 and Csm1. Here, we present evidence that changes in histone H3 lysine methylation levels distinctly regulate rDNA silencing by recruiting different silencing proteins to rDNA, thereby contributing to rDNA silencing and nucleolar organization in yeast.

Results: We found that Lys4, Lys79, and Lys36 methylation within histone H3 acts as a bivalent marker for the regulation of rDNA recombination and RENT complex-mediated rDNA silencing, both of which are Sir2-dependent pathways. By contrast, we found that Jhd2, an evolutionarily conserved JARID1 family H3 Lys4 demethylase, effects all states of methylated H3K4 within the NTS regions of rDNA and that its activity is required for the regulation of rDNA silencing in a Sir2-independent manner. In this context, Jhd2 regulates rDNA recombination through the Tof2/Csm1/Lrs4 pathway and prevents excessive recruitment of Tof2, Csm1/Lrs4 and condensin subunits to the replication fork barrier (RFB) site within the NTS1 region. Our FISH analyses further demonstrate that the demethylase activity of Jhd2 regulates mitotic rDNA condensation and that JHD2-deficient cells contain the mostly hypercondensed rDNA mislocalized away from the nuclear periphery.

Conclusions: Our results show that yeast Jhd2, which demethylates histone H3 Lys4 near the rDNA locus, regulates rDNA repeat stability and rDNA silencing in a Sir2-independent manner by maintaining Csm1/Lrs4 and condensin association with rDNA regions during mitosis. These data suggest that Jhd2-mediated alleviation of excessive Csm1/Lrs4 or condensin at the NTS1 region of rDNA is required for the integrity of rDNA repeats and proper rDNA silencing during mitosis.

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Loss of Jhd2 enhances rDNA silencing at NTS1, but not NTS2, in a Sir2-independent manner. (A) Schematic of an rDNA unit with the position of the mURA3 reporters inserted into NTS1 or NTS2 (see Figure 2A for features). (B, C)URA3-based rDNA silencing assays at either NTS1 or NTS2. WT and the indicated deletion strains in the DMY2798 (leu2::mURA3), DMY2804 (RDN1-NTS1::mURA3) or DMY2800 (RDN1-NTS2::mURA3) background were used. Chr, chromosome; WT, wild type.
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Fig4: Loss of Jhd2 enhances rDNA silencing at NTS1, but not NTS2, in a Sir2-independent manner. (A) Schematic of an rDNA unit with the position of the mURA3 reporters inserted into NTS1 or NTS2 (see Figure 2A for features). (B, C)URA3-based rDNA silencing assays at either NTS1 or NTS2. WT and the indicated deletion strains in the DMY2798 (leu2::mURA3), DMY2804 (RDN1-NTS1::mURA3) or DMY2800 (RDN1-NTS2::mURA3) background were used. Chr, chromosome; WT, wild type.

Mentions: To establish further the role of Jhd2 in rDNA silencing, we used strains carrying an mURA3 reporter gene integrated into one of three sites: outside the rDNA array at the LEU2 gene or inside the rDNA unit at one of two loci with a strong signal for Net1 and Sir2 association (Figure 4A) [13]. Unlike the mURA3 gene outside the rDNA, the mURA3 gene inserted at either NTS1 or NTS2 exhibited strong silencing, which was compromised by sir2Δ in both cases, indicating that silencing at the NTS regions is influenced by Sir2 (Figure 4B, top and middle panes) [13]. The enhanced silencing at NTS2 correlated with increased enrichment of Net1 at chromatin near NTS2 (compare Figures 2C and 4B). By contrast, cells lacking Jhd2 displayed a profound increase in rDNA silencing, predominantly at NTS1, consistent with the ability of Jhd2 to modulate H3K4 methylation primarily at NTS1. Moreover, the sir2Δ jhd2Δ double mutation still significantly increased rDNA silencing at NTS1 to levels seen in jhd2Δ cells and decreased silencing at NTS2 to levels seen in sir2Δ cells. If the enhanced rDNA silencing at NTS1 caused by jhd2Δ was affected by Sir2, then deletion of SIR2 should have at least partially rescued the growth defect observed in jhd2Δ cells (Figure 4B, top and middle panes). Silencing in gis1Δ cells was indistinguishable from that in WT cells. We therefore reasoned that Jhd2 regulates rDNA silencing specifically at the NTS1 region in a Sir2-independent manner.Figure 4


Yeast histone H3 lysine 4 demethylase Jhd2 regulates mitotic rDNA condensation.

Ryu HY, Ahn S - BMC Biol. (2014)

Loss of Jhd2 enhances rDNA silencing at NTS1, but not NTS2, in a Sir2-independent manner. (A) Schematic of an rDNA unit with the position of the mURA3 reporters inserted into NTS1 or NTS2 (see Figure 2A for features). (B, C)URA3-based rDNA silencing assays at either NTS1 or NTS2. WT and the indicated deletion strains in the DMY2798 (leu2::mURA3), DMY2804 (RDN1-NTS1::mURA3) or DMY2800 (RDN1-NTS2::mURA3) background were used. Chr, chromosome; WT, wild type.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4201760&req=5

Fig4: Loss of Jhd2 enhances rDNA silencing at NTS1, but not NTS2, in a Sir2-independent manner. (A) Schematic of an rDNA unit with the position of the mURA3 reporters inserted into NTS1 or NTS2 (see Figure 2A for features). (B, C)URA3-based rDNA silencing assays at either NTS1 or NTS2. WT and the indicated deletion strains in the DMY2798 (leu2::mURA3), DMY2804 (RDN1-NTS1::mURA3) or DMY2800 (RDN1-NTS2::mURA3) background were used. Chr, chromosome; WT, wild type.
Mentions: To establish further the role of Jhd2 in rDNA silencing, we used strains carrying an mURA3 reporter gene integrated into one of three sites: outside the rDNA array at the LEU2 gene or inside the rDNA unit at one of two loci with a strong signal for Net1 and Sir2 association (Figure 4A) [13]. Unlike the mURA3 gene outside the rDNA, the mURA3 gene inserted at either NTS1 or NTS2 exhibited strong silencing, which was compromised by sir2Δ in both cases, indicating that silencing at the NTS regions is influenced by Sir2 (Figure 4B, top and middle panes) [13]. The enhanced silencing at NTS2 correlated with increased enrichment of Net1 at chromatin near NTS2 (compare Figures 2C and 4B). By contrast, cells lacking Jhd2 displayed a profound increase in rDNA silencing, predominantly at NTS1, consistent with the ability of Jhd2 to modulate H3K4 methylation primarily at NTS1. Moreover, the sir2Δ jhd2Δ double mutation still significantly increased rDNA silencing at NTS1 to levels seen in jhd2Δ cells and decreased silencing at NTS2 to levels seen in sir2Δ cells. If the enhanced rDNA silencing at NTS1 caused by jhd2Δ was affected by Sir2, then deletion of SIR2 should have at least partially rescued the growth defect observed in jhd2Δ cells (Figure 4B, top and middle panes). Silencing in gis1Δ cells was indistinguishable from that in WT cells. We therefore reasoned that Jhd2 regulates rDNA silencing specifically at the NTS1 region in a Sir2-independent manner.Figure 4

Bottom Line: We found that Lys4, Lys79, and Lys36 methylation within histone H3 acts as a bivalent marker for the regulation of rDNA recombination and RENT complex-mediated rDNA silencing, both of which are Sir2-dependent pathways.By contrast, we found that Jhd2, an evolutionarily conserved JARID1 family H3 Lys4 demethylase, effects all states of methylated H3K4 within the NTS regions of rDNA and that its activity is required for the regulation of rDNA silencing in a Sir2-independent manner.Our results show that yeast Jhd2, which demethylates histone H3 Lys4 near the rDNA locus, regulates rDNA repeat stability and rDNA silencing in a Sir2-independent manner by maintaining Csm1/Lrs4 and condensin association with rDNA regions during mitosis.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Nucleolar rDNA is tightly associated with silent heterochromatin, which is important for rDNA stability, nucleolar integration, and cellular senescence. Two pathways have been described that lead to rDNA silencing in yeast: 1) the RENT (regulator of nucleolar silencing and telophase exit) complex, which is composed of Net1, Sir2, and Cdc14 and is required for Sir2-dependent rDNA silencing; and 2) the Sir2-independent silencing mechanism, which involves the Tof2 and Tof2-copurified complex, made up of Lrs4 and Csm1. Here, we present evidence that changes in histone H3 lysine methylation levels distinctly regulate rDNA silencing by recruiting different silencing proteins to rDNA, thereby contributing to rDNA silencing and nucleolar organization in yeast.

Results: We found that Lys4, Lys79, and Lys36 methylation within histone H3 acts as a bivalent marker for the regulation of rDNA recombination and RENT complex-mediated rDNA silencing, both of which are Sir2-dependent pathways. By contrast, we found that Jhd2, an evolutionarily conserved JARID1 family H3 Lys4 demethylase, effects all states of methylated H3K4 within the NTS regions of rDNA and that its activity is required for the regulation of rDNA silencing in a Sir2-independent manner. In this context, Jhd2 regulates rDNA recombination through the Tof2/Csm1/Lrs4 pathway and prevents excessive recruitment of Tof2, Csm1/Lrs4 and condensin subunits to the replication fork barrier (RFB) site within the NTS1 region. Our FISH analyses further demonstrate that the demethylase activity of Jhd2 regulates mitotic rDNA condensation and that JHD2-deficient cells contain the mostly hypercondensed rDNA mislocalized away from the nuclear periphery.

Conclusions: Our results show that yeast Jhd2, which demethylates histone H3 Lys4 near the rDNA locus, regulates rDNA repeat stability and rDNA silencing in a Sir2-independent manner by maintaining Csm1/Lrs4 and condensin association with rDNA regions during mitosis. These data suggest that Jhd2-mediated alleviation of excessive Csm1/Lrs4 or condensin at the NTS1 region of rDNA is required for the integrity of rDNA repeats and proper rDNA silencing during mitosis.

Show MeSH
Related in: MedlinePlus